Solid culture medium and method for preparing the same

ABSTRACT

A solid medium having a 10 minute-average water absorption rate of at least 0.05 ml/minute, which is obtainable by a method for producing a solid medium comprising the steps of dissolving components of the solid medium other than solvent water into the solvent water, solidifying the obtained solution, and drying the solidified medium to remove water, wherein water is removed in such an amount that the solid medium after the removal of water should have the 10 minute-average water absorption rate of at least 0.05 ml/minute, and the amount of the solvent water is larger than a prescribed amount by an amount almost equal to the amount of the water to be removed. The solid medium does not cause growth inhibition of microorganisms due to drying, shows a superior water absorption rate to enable application of a larger amount of a sample in a short period of time, and is suitable for quick and accurate measurement tests of microbial numbers.

TECHNICAL FIELD

[0001] The present invention relates to a solid medium used for growthand tests of various microorganisms and so forth and a method forproducing the same.

BACKGROUND ART

[0002] As an example of dried solid media, a dried solid medium isconventionally known which is prepared by lyophilizing a medium pouredinto a plastic dish and solidified (JP 60-19988B, referred to as “Priorart 1” hereinafter).

[0003] Further, there is also a known dried solid medium in the form ofa film, which contains a gelling agent such as agar and is easilysubstantially restored to a previous state before it is formed into filmby adding sterilized water (JP 6-311880A, referred to as “Prior art 2”hereinafter).

[0004] However, these conventional materials have the followingproblems.

[0005] Prior art 1 and Prior art 2 mentioned above are used in order toimprove storage stability of media and the media are dried so that awater content in the compositions of finally obtained solid mediumbecomes 50% or less. Therefore, they have a problem that they require along period of time as long as about 3 hours for full restoration of themedia upon use.

[0006] Further, since the dried solid media obtained according to theaforementioned conventional techniques require a long period of time forrestoration as described above, restoration in a relatively short periodsuch as 1 to 10 minutes results in insufficient restoration. If suchmedia are used for microorganism tests, growth of Staphylococcus aureusATCC 6538P, Bacillus stearothermophilus var. calidolactis NIZO C953etc., which are important bacterial species in microorganism tests offoodstuffs and so forth, is partially inhibited due to the insufficientrestoration of the medium, resulting in inaccurate measurement of thenumber of microorganisms, as demonstrated by the Test Examples mentionedlater.

[0007] That is, the conventional dried solid media have a problem thatthey require a long period of time for restoration, and thus they arenot suitable for quick and accurate measurement tests of microbialnumbers.

[0008] Further, it is known that a solid medium is dried in order toremove excessive moisture on its surface after its production. Sincethis drying process removes a part of solvent water from the solidmedium prepared in a prescribed composition, water content of the mediumupon actual culture of microorganisms should significantly differ fromthe prescribed content in the composition identifying the solid medium.Influences of medium-drying on the culture of microorganisms cannot beignored. For example, it is known that growth of microorganisms issometimes degraded due to drying etc., even when a medium is stored in atest tube with a cotton plug for several weeks. Moreover, uncontrolledremoval of water sometimes makes the composition of solid mediainconstant. Therefore, reproducible and accurate results may not beobtained in culture tests of microorganisms, especially those ofmicroorganisms that are likely to be affected by the water content ofmedium.

DISCLOSURE OF THE INVENTION

[0009] An object of the present invention is to provide a solid mediumwhich shows a superior water absorption rate to enable application of alarge amount of sample in a short period of time and is suitable forquick and accurate measurement tests of microbial numbers and to providea method for producing the same.

[0010] The inventors of the present invention assiduously studied inview of the aforementioned problems of the prior art. As a result, theyobtained a solid medium by dissolving components of the solid mediuminto the solvent water whose amount is larger than a prescribed amount,solidifying the obtained solution and drying the solidified medium toremove water in an amount almost equal to the excessive amount of thesolvent water. They have found that the medium does not cause growthinhibition of microorganisms due to drying. They have also found that itshows superior water absorption rate to enable application of a largeamount of a sample in a short period of time and is suitable for quickand accurate measurement tests of microbial numbers. Thus, theyaccomplished the present invention.

[0011] The present invention provides a solid medium having a 10minute-average water absorption rate of at least 0.05 ml/minute (alsoreferred to as the “solid medium of the present invention” hereinafter),which is obtainable by a method for producing a solid medium comprisingthe steps of dissolving components of the solid medium other thansolvent water into the solvent water, solidifying the obtained solution,and drying the solidified medium to remove water, wherein water isremoved in such an amount that the solid medium after the removal ofwater should have the 10 minute-average water absorption rate of atleast 0.05 ml/minute, and the amount of the solvent water is larger thana prescribed amount by an amount almost equal to the amount of the waterto be removed.

[0012] In the solid medium of the present invention, the amount of thewater to be removed is preferably at least 5% of the solvent water, morepreferably at least 30% of the solvent water.

[0013] The solid medium of the present invention preferably has a watercontent of at least 90%.

[0014] The present invention also provides a method for producing asolid medium comprising the steps of dissolving components of the solidmedium other than solvent water into the solvent water, solidifying theobtained solution and drying the solidified medium to remove water,wherein water is removed in such an amount that the solid medium afterthe removal of water should have a 10 minute-average water absorptionrate of at least 0.05 ml/minute, and the amount of the solvent water islarger than a prescribed amount by an amount almost equal to the amountof the water to be removed.

[0015] In the production method of the present invention, the amount ofthe water to be removed is preferably at least 5% of the solvent water,more preferably at least 30% of the solvent water.

[0016] In the present specification, a solid medium means a medium thatis in a solid state at the time of use, for example, a plate medium,slant medium and so forth, which are solidified with a gelling agentsuch as agar.

[0017] A medium is generally identified (defined) by its composition.Thus, the prescribed amount of solvent water is an amount of water insuch a composition used for the identification of the medium. Theprescribed amount of solvent water means an amount of water added inorder to dissolve each component of the solid medium other than solventwater, and when a component of the solid medium is specified as anaqueous solution, the amount of water in this component is not included.

[0018] The 10 minute-average water absorption rate is calculated inaccordance with the following equation by using A and B measured asfollows. Total weight (B) of a sample plate (circular shape, outerdiameter: 9 cm, inner diameter: 8.6 cm (area: 58.1 cm²), amount ofmedium: 15 g) is measured, and water absorption is started by adding 10ml of ion-exchanged water onto the plate medium. After 10 minutes, waterthat has not been absorbed is discarded, moisture on a dish wall iswiped off, and the total weight (A) of the sample plate after the waterabsorption is measured. The measurement is performed at 25° C. When thearea of the sample plate differs from the aforementioned area, the testis performed by using ion exchanged water in an amount of 10 ml perabove-defined area, and the result is converted into a water absorptionrate per above-mentioned area.

[0019] Water absorption rate (ml/minute)=(A−B)/10

[0020] The amount of water removed by drying is calculated by measuringthe weight loss due to drying. That is, the weight loss due to drying,i.e., so-called water loss (c), can be obtained from the medium weightbefore the drying (a) and the medium weight after the drying (b) inaccordance with an equation of a−b=c. Then, a ratio of the removedwater, i.e., percentage of water removed by drying (e, %) can beobtained from the total amount of added solvent water (d) and theaforementioned weight loss due to drying (c) in accordance with thefollowing equation.

e(%)=c/d×100

[0021] A water content of solid medium means a percentage of watercontent (%) defined as (amount of water in solid medium)/(amount ofsolid content+amount of water)×100. Here, both the amount of water andthe solid content are represented in weight.

Best Mode for Carrying out the Invention

[0022] A medium is a nutriment for growing or proliferatingmicroorganisms such as bacteria, yeasts and molds, and it is also theirgrowing environment. A medium usually contains, as medium components, asaccharide such as glucose and lactose, a nitrogen source such as aminoacids, peptone, nitrates and ammonium salts, inorganic salts such asthose of potassium, phosphorus and magnesium, a growth factor such asvitamins and so forth. Media are roughly classified into liquid media inwhich medium components are simply dissolved in solvent water and solidmedia solidified by addition of a gelling agent into liquid media. Thepresent invention provides a solid medium, which is, as defined above, amedium (culture medium) in a solid state at the time of use, forexample, a plate medium, slant medium or the like, produced bysolidifying with a gelling agent such as agar.

[0023] Examples of the gelling agent include agar, gelatin, gellan gum,carrageenan and so forth.

[0024] Specific examples of the composition of the solid medium of thepresent invention include the compositions of the followings: thenutrient agar medium, the standard agar medium, the deoxycholate agarmedium, the E.M.B. medium, the Endo medium, the plate count agar mediumwith B.C.P., the mannitol salt agar with egg yolk, the potato dextroseagar medium, the Violet red bile lactose (VRBL) agar medium, the yeastextract-glucose-chloramphenicol agar medium, the Acidified MRS medium,Medium M17, the Baird-Parker agar medium (ETGP agar medium) and soforth, which are defined in microorganism test methods described in theJapanese Food Sanitation Law, the Japan Pharmacopoeia, the InternationalDiary Federation Standard (IDF STANDARD) and so forth [“NyuseihinShikenhou Chukai (Commentary of Test Methods of Dairy Products)”, Ed. bythe Pharmaceutical Society of Japan, pp.111-125, Apr. 10, 1990, KaneharaShuppan Co., Ltd. (Reference 1); “IDF STANDARD (Revised Version of1991)”, p.306, p.470, pp.645-647, Dec. 25, 1991, published byInternational Dairy Federation of Japan (Reference 2); “Shin SaikinBaichi-gaku Koza Ge II (Lecture of Culture Medium Science for Bacteria,Vol. 2, II)”, Second edition, Editorship: Sakazaki Toshikazu, pp.62-63,Aug. 15, 1996, Kindai Shuppan Co., Ltd. (Reference 3)).

[0025] For reference, the prescribed amounts of respective componentscontained in the aforementioned solid media described in Reference 1 arementioned below.

[0026] Prescribed amounts of components in the solid medium compositionof the nutrient agar medium (pH 7.0 to 7.4) are 5 g of meat extract, 10g of peptone, 1 to 2 g of sodium chloride (NaCl), 12 to 15 g of agar and1000 ml (1 liter) of purified water (solvent water ).

[0027] Prescribed amounts of components in the solid medium compositionof the standard agar medium (pH 6.8 to 7.2) are 2.5 g of yeast extract,5 g of peptone, 1 g of glucose, 15 g of agar and 1000 ml (1 liter) ofpurified water (solvent water).

[0028] Prescribed amounts of each component in the solid mediumcomposition of the deoxycholate agar medium (pH 7.0 to 7.4) are 10 g ofpeptone, 10 g of lactose, 1 g of sodium deoxycholate, 5 g of sodiumchloride (NaCl), 2 g of K₂HPO₄, 0.033 g of Neutral Red, 2 g of ferricammonium citrate, 15 g of agar and 1000 ml (1 liter) of purified water(solvent water).

[0029] Prescribed amounts of components in the solid medium compositionof the E.M.B. medium (pH 6.6 to 7.0) are 10 g of peptone, 10 g oflactose, 2 g of K₂HPO₄, 0.4 g of Eosin Y, 0.065 g of Methylene Blue, 18g of agar and 1000 ml (1 liter) of purified water (solvent water).

[0030] Prescribed amounts of components in the solid medium compositionof the Endo medium (pH 7.0 to 7.4) are 10 g of peptone, 3 g of meatextract, 10 g of lactose, 1.6 g of Na₂SO₃, 0.1 g of Basic Fuchsine, 15 gof agar and 1000 ml (1 liter) of purified water (solvent water).

[0031] Prescribed amounts of components in the solid medium compositionof the plate count agar medium with B.C.P. (pH 6.0 to 7.0) are 2.5 g ofyeast extract, 5 g of peptone, 1 g of glucose, 1 g of Tween 80, 0.1 g ofL-cysteine, 0.06 g of Bromocresol Purple, 15 g of agar and 1000 ml (1liter) of purified water (solvent water).

[0032] Prescribed amounts of components in the solid medium compositionof the mannitol salt agar with egg yolk [pH 7.2 to 7.6, where 50 to 60mlof fresh egg yolk solution (obtained by dissolving egg yolk into thesame amount of physiological saline) is added after sterilization withhigh pressure steam and before pouring the medium into plate] are 2.5 gof meat extract, 10 g of peptone, 10 g of mannitol, 75 g of sodiumchloride (NaCl), 0.025 g of Phenol Red, 15 g of agar and 1000 ml (1liter) of purified water (solvent water).

[0033] Prescribed amounts of components in the solid medium compositionof the potato dextrose agar (pH 5.6 to 5.7) are 200 g of potatoinfusion, 20 g of glucose, 15 g of agar and 1000 ml (1 liter) ofpurified water (solvent water).

[0034] Prescribed amounts of respective components contained in thesolid media described in Reference 2 are mentioned hereafter.

[0035] Prescribed amounts of components in the solid medium compositionof the Violet red bile salt lactose (VRBL) agar medium (pH 7.4±0.1) are7 g of peptone, 3 g of yeast extract, 10 g of lactose (C₁₂H₂₂O₁₁.H₂O), 5g of sodium chloride (NaCl), 1.5 g of bile acid salt, 0.03 g of NeutralRed, 0.002 g of Crystal Violet, 12 to 18 g of agar and 1000 ml (1 liter)of water (solvent water).

[0036] Prescribed amounts of components in the solid medium compositionof the yeast extract-glucose-chloramphenicol agar medium (pH 6.6) are 5g of yeast extract, 20 g of glucose (C₆H₁₂O₆), 0.1 g of chloramphenicol(C₁₁H₁₂Cl₂N₂O₅) or oxytetracycline (C₂₂H₃₀N₂O₁₁), 12 to 15 g of agar and1000 ml (1 liter) of water (solvent water).

[0037] Prescribed amounts of components in the solid medium compositionof the acidified MRS medium (adjusted to pH 5.4 with acetic acid) are 10g of Peptone 1 (trypsin-digested product of casein), 10 g of meatextract, 5 g of yeast extract (powder), 20 g of glucose (C₆H₁₂O₆), 1 mlof Tween 80 (sorbitan mono-oleate), 2 g of dipotassiumhydrogenorthophosphate (K₂HPO₄), 5 g of sodium acetate trihydrate(CH₃CO₂Na.3H₂O), 2 g of diammonium citrate (C₆H₆O₇(NH₄)₂), 0.2 gmagnesium sulfate heptahydrate (MgSO₄.7H₂O), 0.05 g of manganese sulfatetetrahydrate (MnSO₄.4H₂O), 9 to 18 g of agar and 1000 ml (1 liter) ofwater (solvent water).

[0038] Prescribed amounts of components in the solid medium compositionof Medium M17 [pH 7.1 to 7.2, where lactose (C₁₂H₂₂O₁₁) is added as asterilized 10% lactose (C₁₂H₂₂O₁₁) aqueous solution after sterilizationwith high pressure steam and before pouring the medium into plate] are2.50 g of Peptone 1 (trypsin-digested product of casein), 2.50 g ofPeptone 2 (pepsin-digested product of meat), 5.00 g of Peptone 3 (papain-digested product of soybean), 2.50 g of yeast extract (powder), 5.00 gof meat extract, 19.00 g of β-glycerophosphoric acid salt (disodiumsalt, C₆H₇O₆PNa₂), 0.25 g of magnesium sulfate heptahydrate(MgSO₄.7H₂O), 0.50 g of ascorbic acid (C₆H₈O₆), 5 g of lactose(C₁₂H₂₂O₁₁), 9 to 18 g of agar and 1000 ml (1 liter) of water (solventwater).

[0039] Prescribed amounts of components contained in the solid mediumdescribed in Reference 3 are mentioned hereafter.

[0040] Prescribed amounts of components in the solid medium compositionof the Baird-Parker agar medium (ETGP agar medium, pH 6.8, where10 mlof1% potassium tellurite aqueous solution and 50 ml of 50% egg yolkemulsion are added after sterilization with high pressure steam andbefore pouring the medium into plate) are 10 g of peptone, 5 g of meatextract, 1 g of yeast extract, 5 g of lithium chloride, 12 g of glycine,10 g of sodium pyruvate, 17 g of agar and 1000 ml (1 liter) of purifiedwater (solvent water).

[0041] The method for producing a solid medium of the present inventionis explained hereafter.

[0042] In the method for producing a solid medium of the presentinvention, dissolution of components of the solid medium into solventwater and solidification of the solution can be performed in the samemanner as in the production of usual solid media except that the amountof the solvent water is larger than its prescribed amount and that wateris removed by drying after the solidification. For example, prescribedamounts of medium components described in literature etc. or newlyidentified as a solid medium composition (defined) can be dissolved insolvent water; after adjusting pH when required, a gelling agent can beadded to the solution; and the mixture can be heated to dissolve thegelling agent, sterilized, poured into a vessel like a dish andsolidified.

[0043] The amount of water to be removed is such an amount that the 10minute-average water absorption rate of the solid medium should becomeat least 0.05 ml/minute after the removal of water. Although this amountmay vary depending on the medium composition (especially depending onthe gelling agent), it can be determined by producing media containingsolvent water in various excessive amounts and measuring their 10minute-average water absorption rates in such a manner as described inTest Example 3 mentioned later.

[0044] The amount of water to be removed is preferably at least 5% ofthe solvent water, more preferably at least 30% of the solvent water.

[0045] When water is removed in above-mentioned manner from a solidmedium prepared by using a prescribed amount of solvent water, theamount of water contained in the solid medium may be significantlyreduced after the removal of water, resulting in growth inhibition ofmicroorganisms due to drying. According to the present invention, theamount of solvent water in the solid medium is determined beforehand tobe larger than the prescribed one, so that water is contained in thesolid medium in an amount corresponding to the prescribed amount afterthe removal of water. Therefore, the amount of solvent water is largerthan the prescribed amount by an amount almost equal to that of water tobe removed. The amount almost equal to that of water to be removedherein means the amount at which, the amount obtained by subtracting theamount of water to be removed from the amount of the total added solventwater including the prescribed amount and the additional amount ofwater, should become substantially equal to (usually, 97 to 103%) theprescribed amount of water.

[0046] Usually, the additional amount of the solvent water is 5 to 150%when the amount of solvent water (prescribed amount of solvent water) ina composition described literature etc., or in a composition newlyidentified as a solid medium composition, is taken as 100%. This amountof solvent water is formulated in addition to the prescribed amount ofwater. This enables the production of the solid medium of the presentinvention which contains a prescribed amount of water and has asufficient water absorption rate, as an end product obtained by removingsolvent water by drying.

[0047] When an agar is used as the gelling agent, the concentration ofthe added agar is preferably 1.0 to 3% (by weight) when theaforementioned prescribed amount of solvent water is taken as 100%.Further, the heating for dissolution is preferably performed at atemperature of 100° C. or higher, since agar is fully dissolved at sucha temperature. If the heating is performed at 121° C. for 15 minutes ormore, sterilization can also be attained simultaneously with thedissolution. Sterilization may be performed in a conventional manner byusing high pressure steam sterilization method or the like.

[0048] When dishes are used as the vessels, the solid medium ispreferably poured into each dish in such an amount that the amount ofthe solid medium in each dish after the removal of water by dryingshould become 10 to 30 ml.

[0049] Examples of the drying method to remove water from the mediuminclude reduced pressure drying method, vacuum evaporation method, warmair drying method, infrared drying method, high frequency drying methodand so forth. However, the method is not limited to these.

[0050] The solid medium of the present invention has 10 minute-averagewater absorption rate of at least 0.05 ml/minute according to thedefinition mentioned above. If the solid medium shows water absorptionof at least 0.5 ml for 10 minutes per one plate of the standard sizedefined above, it becomes possible to apply a large amount of a sampleonto the solid medium in a short period of time. A solid medium showingthe 10 minute-average water absorption rate of at least 0.05 ml/minutecan be prepared by drying the medium to remove such an appropriateamount of water as described above. The amount of medium per exposedarea (area in contact with air) of the solid medium can be such anamount that the 10 minute-average water absorption rate in theaforementioned range can be obtained. If the amount of the medium is toosmall, a sufficient 10 minute-average water absorption rate may not beobtained.

[0051] Since the solid medium of the present invention contains solventwater in an amount almost equal to the prescribed amount after theremoval of water, growth inhibition of microorganisms resulting fromdrying is not observed on it. This is because a prescribed amount ofsolvent water in a composition identifying a solid medium is determinedso that a microorganism to be cultured can grow. Alternatively, it isbecause a medium having a composition that does not cause growthinhibition of a microorganism to be cultured is selected.

[0052] The solid medium preferably has a water content of at least 90%.Such water content enables culture of microorganism requiring high watercontent. For example, since growth of a microorganism such asStaphylococcus aureus ATCC 6538P is almost completely inhibited in watercontent lower than 90% as demonstrated in the Test Examples mentionedlater, it is necessary to keep water content of at least 90% in thesolid medium in order to culture the microorganism.

[0053] Furthermore, the solid medium of the present invention issuitable for culturing microorganisms that are relatively weak todryness, but important for microorganism tests of foodstuffs and soforth, such as Staphylococcus aureus ATCC 6538P and Bacillusstearothermophilus var. calidolactis NIZO C953, without causing anygrowth inhibition thereof. As described above, the solid medium of thepresent invention is dried in a state that it contains solvent water inan amount larger than the prescribed amount. Therefore, it maintains asufficient water absorption rate and does not cause growth inhibitiondue to drying.

[0054] In order to make it much easier to understand the method forproducing a solid medium of the present invention, it is explained byexemplifying a method for producing the standard agar medium describedin Reference 1 mentioned above.

[0055] Namely, prescribed amounts of components in the solid mediumcomposition of the standard agar medium are 2.5 g of yeast extract, 5 gof peptone, 1 g of glucose, 15 g of agar and 1000 ml of purified water(solvent water), as described in Reference 1.

[0056] Therefore, the component of 2.5 g of yeast extract, 5 g ofpeptone and 1 g of glucose are dissolved in solvent water whose amountis preferably 5 to 150% (50 to 1500 ml) larger than the prescribedamount (1000 ml) to obtain a dissolved solution in an amount from about1050 ml to 2500 ml. The dissolved solution is adjusted to pH 6.8 to 7.2.15 g of agar is added to the solution and the mixture is heated at 121°C. for 15 minutes by the high pressure steam sterilization method todissolve and sterilize simultaneously.

[0057] The sterilized medium is aseptically poured into dishes in anamount of 16 to 38 g per dish, and solidified by cooling. The solidifiedmedium is then dried by the reduced pressure drying method under apressure of 10³ Pa using a dryer (LABCONCO) to remove solvent water.

[0058] By the aforementioned method to remove water by drying, theadditional amount of water is removed to produce a solid medium whichhas the 10 minute-average water absorption rate of at least 0.05ml/minute.

[0059] The solid medium produced by the aforementioned method does notcause growth inhibition of microorganisms resulting from drying.Moreover, it shows a superior water absorption rate and enablesapplication of a large amount of a sample in a short period of time andis suitable for quick and accurate measurement tests of microbialnumbers.

EXAMPLES

[0060] The present invention is explained in more detail by thefollowing examples. However, the present invention is not limited to thefollowing examples.

Example 1

[0061]2.5 g of yeast extract (Oriental Yeast), 5 g of peptone (Difco)and 1 g of glucose (Wako Pure Chemical Industries) were dissolved in1200 ml of solvent water which contains 20% (200 ml) excess water inaddition to the prescribed amount of solvent water (purified water, 1000ml), which was taken as 100%, in a known solid medium composition toobtain about 1200 ml of a dissolved solution. The solution was adjustedto pH 7.2 by using 0.1 mol/l sodium hydroxide solution. 15 g of agar(Ina Shokuhin Kogyo) was added to the solution and the mixtures washeated at 121° C. for 15 minutes by using an autoclave (Iwatate IryoKikai Seisakusho) to attain dissolution and sterilization at the sametime.

[0062] The sterilized solid medium was poured into each plastic dishhaving a diameter of 9 cm (Eiken Kizai) in an amount of 18 g per dish ina sterile room, solidified by cooling and dried at a pressure of 10³ Paaccording to the reduced pressure drying method using a drier (LABCONCO)to remove 3 ml of water. As a result, solid medium plates eachcontaining 15 g of the medium were obtained.

[0063] The aforementioned removal of water corresponded to removal ofthe total amount of the solvent water (200 ml) formulated in addition tothe prescribed amount of water, i.e., removal of about 17% (200 ml) ofthe total amount (1200 ml) of the added solvent water (purified water),which was taken as 100%, by drying.

[0064] Thus, the water content of the solid medium has been adjusted toabout 98% by the aforementioned removal of water.

[0065] The solid medium (the nutrient agar medium) plates produced asdescribed above were tested by the method described later, and they werefound to have the 10 minute-average water absorption rate of 0.15ml/minute. It has been found that the medium is a solid medium capableof application of a large amount of a sample in a short period of timeand which does not show growth inhibition caused by drying, whereby itis suitable for quick and accurate measurement tests of microbialnumbers.

Example 2

[0066]2.5 g of meat extract (Merck),10 g of peptone (Difco), 75 g ofsodium chloride (Wako Pure Chemical Industries), 10 g of mannitol (WakoPure Chemical Industries) and 0.025 g of Phenol Red (Wako Pure ChemicalIndustries) were dissolved in 1066 ml of solvent water which contains6.6% (66 ml) excess water in addition to the prescribed amount ofsolvent water (purified water, 1000 ml) taken as 100%, in a known solidmedium composition, to obtain about 1066 ml of a dissolved solution. Thedissolved solution was adjusted to pH 7.4 by using 0.1 mol/l sodiumhydroxide solution. 15 g of agar (Ina Shokuhin Kogyo) was added to thesolution and the mixture was heated at 121° C. for 15 minutes by usingan autoclave (Iwatate Iryo Kikai Seisakusho) to attain dissolution andsterilization at the same time.

[0067] The sterilized solid medium was poured into each plastic dishhaving a diameter of 9 cm (Eiken Kizai) in an amount of 16 g per dish ina sterile room, solidified by cooling and dried at a pressure of 10³ Paaccording to the reduced pressure drying method using a drier (LABCONCO)to remove about 1 ml of solvent water. As a result, solid medium plateseach containing 15 g of the medium were obtained.

[0068] The aforementioned removal of water corresponded to removal ofthe total amount of the solvent water (66 ml) formulated in addition tothe prescribed amount of water, i.e., removal of about 6% (66 ml) of thetotal amount (1066 ml) of the added solvent water (purified water) takenas 100%, was removed by drying.

[0069] Therefore, the water content of the solid medium has beenadjusted to about 90% by the aforementioned removal of water.

[0070] The solid medium (the mannitol salt agar with egg yolk) platesproduced as described above were tested by the method described later,and they were found to have the 10 minute-average water absorption rateof 0.07 ml/minute. It has been found that the medium is a solid mediumwhich enables application of a large amount of a sample in a shortperiod of time and which does not show microbial growth inhibition ofStaphyiococcus bacteria caused by drying, whereby it is suitable forquick and accurate measurement tests of microbial numbers.

Example 3

[0071] A solid medium having a novel composition was prepared bymodifying a composition of a known solid medium. 10 g of peptone(Difco), 5 g of meat extract (Merck) and 2 g of sodium chloride (WakoPure Chemical Industries) were dissolved in 1200 ml of solvent water,which contains 33.3% (300 ml) of excess water in addition to aprescribed amount of 900 ml, taken as 100%, which was modified to a lessamount than the prescribed amount of solvent water (purified water, 1000ml) in a known solid medium composition, to obtain about 1200 ml of adissolved solution. The dissolved solution was adjusted to pH 7.0 byusing 0.1 mol/l sodium hydroxide solution. 15 g of agar (Ina ShokuhinKogyo) was added to the solution and the mixture was heated at 121° C.for 15 minutes by using an autoclave (Iwatate Iryo Kikai Seisakusho) toattain dissolution and sterilization at the same time.

[0072] The sterilized solid medium was poured into each plastic dishhaving a diameter of 9 cm (Eiken Kizai) in an amount of 20 g per dish ina sterile room, solidified by cooling and dried at a pressure of 10³ Paaccording to the reduced pressure drying method using a drier (LABCONCO)to remove 5 ml of solvent water. As a result, solid medium plates eachcontaining 15 g of the medium were obtained.

[0073] The aforementioned removal water corresponded to removal of thetotal amount (300 ml) of the water formulated in addition to theprescribed amount of water, i.e., removal of about 25% (300 ml) of totalamount (1200 ml) of the added solvent water(purified water), taken as100%, by drying.

[0074] Thus, the water content of the solid medium has been adjusted toabout 96% by the aforementioned removal of water.

[0075] The solid medium (nutrient agar medium) plates produced asdescribed above were tested by the method described later, and they werefound to have the 10 minute-average water absorption rate of 0.17ml/minute. It has been found that the medium is a solid medium whichenables application of a large amount of sample in a short period oftime and which does not show microbial growth inhibition caused bydrying, whereby it is suitable for quick and accurate measurement testsof microbial numbers.

Test Example 1

[0076] This test was performed to compare the present invention with theprior art by using results of microbial growth test as indexes.

[0077] (1) Preparation of Samples

[0078] Each of the following three kinds of samples was prepared inquintuplicate.

[0079] Sample 1: Solid medium produced in the same manner as in Example1 of the present invention.

[0080] Sample 2: Solid medium produced in the same manner as in Example1 of Prior Art 1 except that the type of the medium was changed to thenutrient agar medium, 15 ml of the medium was poured into each dish, 15ml of water for restoration (sterilized water) was used, the solidmedium was restored for 5 minutes and excessive water for restoration(free water) was discarded by decantation.

[0081] Sample 3: Solid medium produced in the same manner as in Example1 of Prior Art 2 except that the type of the medium was changed to thenutrient agar medium, 15 ml of the medium was poured into each dish, 15ml of water for restoration (sterilized water) was used, the solidmedium was restored for 5 minutes and excessive water for restoration(free water) was discarded by decantation.

[0082] (2) Test Method

[0083] Microbial growth on each sample was examined by the followingtest method.

[0084] As test strains, Staphylococcus aureus ATCC 6538P obtained fromthe American Type Culture Collection, which is a depository ofmicroorganisms, and Bacillus stearothermophilus var. calidolactis NIZOC953 obtained from the Netherlands Institute for Dairy Research(Nederlands Instituut voor Zuivelonderzoek, NIZO) were used.

[0085] Such a dilution of each test strain was prepared that, when 0.1ml of the dilution was applied onto one standard agar medium plateprepared by using solvent water in the prescribed amount defined in theknown solid medium composition, i.e., using solvent water in theprescribed amount defined in the known solid medium composition as itwas, without any drying process and incubated at 37° C. for 48 hours,100 colonies of the test strain should be obtained.

[0086] The solid medium composition of the standard agar medium iscomposed of 2.5 g of yeast extract, 5 g of peptone, 1 g of glucose, 15 gof agar and 1000 ml (prescribed amount of solvent water) of purifiedwater.

[0087] The dilution of test strain in an amount of 0.1 ml was appliedonto one plate of each sample and incubated at 37° C. for 48 hours, andnumber of appeared colonies was determined by visual inspection. Thetest was performed in quintuplicate, and an average of the numbers ofcolonies was calculated.

[0088] (3) Test Results

[0089] The results of this test are as shown in Table 1. As clearly seenfrom the results shown in Table 1, it was found that Sample 1 accordingto the present invention is superior to Samples 2 and 3 according toprior art, since Staphylococcus aureus ATCC 6538P and Bacillusstearothermophilus var. calidolactis NIZO C953, which are importantbacteria for microorganism tests of foodstuffs and so forth, could bedetected without any growth inhibition and microbial numbers could bemeasured accurately and quickly with Sample 1.

[0090] Based on the above results, it was found that the dried solidmedia of the conventional techniques required a long period of time forrestoration and thus they were not suitable for quick and accuratemeasurement tests of microbial numbers.

[0091] In addition, when the test was repeated by changing the type ofmedium, the amount of medium poured into each dish, the amount of water(sterilized water) for restoration of dried solid medium and therestoration time of dried solid medium (within the range of 1 to 10minutes), almost similar results were obtained. TABLE 1 Colony number ofColony number of Bacillus Sample Staphylococcus aureusstearothermophilus NIZO No. ATCC 6538P C953 1 102 98 2 35 38 3 40 53

Test Example 2

[0092] This test was performed in order to determine appropriate watercontent in solid medium by using results of microbial growth test asindexes.

[0093] (1) Preparation of Samples

[0094] Each of the following two kinds of samples was prepared inquintuplicate.

[0095] Sample 4: Solid medium prepared in the same manner as in Example2 of the present invention except that solvent water was removed bydrying so that the water content of the solid medium should become 90%

[0096] Sample 5: Solid medium prepared in the same manner as in Example2 of the present invention except that solvent water was removed bydrying so that the water content of the solid medium should become 80%

[0097] (2) Test Method

[0098] Microbial growth on each sample was examined in the same manneras the test method of Test Example 1 mentioned above except that onlythe Staphylococcus aureus ATCC 6538P mentioned above was used as a teststrain.

[0099] Change of water content in the solid medium by drying wasmonitored in terms of weight loss caused by drying. That is, the weightloss by drying, i.e., so-called water loss (c), was obtained from weightof medium before drying (a) and weight of medium after drying (b) inaccordance with the equation of a−b=c. Then, content of remaining waterwas obtained by subtracting the above water loss (c) from the weight ofthe total added solvent water. The water content after drying wasobtained based on the definitions of the remaining water content and theaforementioned water content to monitor the change of water content ofthe solid medium by drying.

[0100] The solid medium composition of the mannitol sodium chloride agarmedium is composed of 2.5 g of meat extract, 10 g of peptone, 75 g ofsodium chloride, 10 g of mannitol, 0.025 g of Phenol Red, 15 g of agarand 1000 ml (prescribed amount of solvent water) of purified water.

[0101] (3) Test Results

[0102] The results of this test are as shown in Table 2. As clearly seenfrom the results shown in Table 2, when the water content of the solidmedium was 80%, so-called less than 90%, growth of Staphylococcus aureusATCC 6538P was almost completely inhibited, which made it impossible tocount the microbial number, thus it was found that a water content of atleast 90% was necessary to measure the numbers accurately.

[0103] In addition, when the test was repeated by changing the type ofmedium and the water content variously, almost similar results wereobtained. TABLE 2 Colony number of Staphylococcus aureus ATCC Sample No.6538P 4 97 5 0

Test Example 3

[0104] This test was performed by using the results of absorption ratetest as indexes in order to determine an appropriate amount of solventwater to be removed (%) when an amount of solvent water described as asolid medium composition in literature and so forth was taken as 100%.

[0105] (1) Preparation of Samples

[0106] Each of the following four kinds of samples was prepared inquintuplicate.

[0107] Sample 6: Solid medium prepared in the same manner as in Example1 of the present invention except that solvent water was not formulatedin addition to the prescribed amount and removal of solvent water bydrying was not performed

[0108] Sample 7: Solid medium prepared in the same manner as in Example1 of the present invention except that solvent water was additionallyformulated by 2.5% of the prescribed amount and 2.5% of the total amountof solvent water was removed by drying.

[0109] Sample 8: Solid medium prepared in the same manner as in Example1 of the present invention except that solvent water was additionallyformulated by 5% of the prescribed amount and 5% of the total amount ofsolvent water was removed by drying.

[0110] Sample 9: Solid medium prepared in the same manner as in Example1 of the present invention except that solvent water was additionallyformulated by 40% of the prescribed amount and 30% of the total amountof solvent water was removed by drying.

[0111] (2) Test Method

[0112] The water absorption rate of each sample was determined by thefollowing test method. The determination was performed at 25° C.

[0113] The total weight of sample plate (B) was measured, then 10 ml ofion exchanged water was added onto a plate medium (circular shape, outerdiameter 9 cm. inner diameter: 8.6 cm (area: 58.1 cm²), amount ofmedium: 15 g) to start water absorption; water not absorbed wasdiscarded when 10 minutes passed; moisture on a dish wall was wiped off;and the total weight of the sample plate after water absorption (A) wasmeasured. Then, the 10 minute-average water absorption rate wascalculated in accordance with the following equation:

Water absorption rate (ml/minute)=(A−B)/10

[0114] Further, percentage of solvent water removed by drying (e) wasobtained from the total amount of formulated solvent water (d) which wastaken as 100%, and the water loss (c) monitored and calculated in thesame manner as in Test Example 2 in accordance with the followingequation:

e(%)=c/d×100

(3) Test Results

[0115] The results of this test are as shown in Table 3. As clearly seenfrom the results shown in Table 3, when at least 5% of solvent water wasremoved by drying the medium, the water absorption rate became at least0.05 ml/minute, and therefore it became possible to apply a large amountof a sample in a short period of time. Thus, it was found that it isnecessary to remove at least 5% of solvent water for quick measurementtests of microbial numbers. Further, when at least 30% of solvent waterwas removed, the water absorption rate became at least 0.2 ml/minute,and therefore it became possible to apply a larger amount of the samplein a short period of time. Thus, it was found that it is preferable toremove at least 30% of solvent water for quick measurement tests ofmicrobial numbers.

[0116] In addition, when the test was repeated by changing the type ofmedium variously, almost similar results were obtained. TABLE 3 10minute-average water Sample No. absorption rate (ml/minute) 6 0.02 70.04 8 0.06 9 0.2

Industrial Applicability

[0117] The advantages of the present invention are as follows.

[0118] 1) The medium of the present invention, which is prepared by theproduction method of the present invention, shows a superior waterabsorption rate, to enable application of a large amount of sample in ashort period of time, and is suitable for quick measurement tests ofmicrobial numbers.

[0119] 2) Since the solid medium of the present invention, which isprepared by the production method of the present invention, enablesapplication of a large amount of sample, it provides high accuracy formicrobial detection and therefore it is suitable for accuratemeasurement tests of microbial numbers.

[0120] 3) Since the solid medium of the present invention, which isprepared by the production method of the present invention, does notshow growth inhibition due to drying, it is suitable for accuratemeasurement tests of microbial numbers.

[0121] 4) A solid medium with a superior water absorption rate whichenables application of a large amount of sample in a short period oftime and suitable for quick and accurate measurement tests of microbialnumbers can be prepared by the production method of the presentinvention.

What is claimed is:
 1. A solid medium having a 10 minute-average waterabsorption rate of at least 0.05 ml/minute, which is obtainable by amethod for producing a solid medium comprising the steps of dissolvingcomponents of the solid medium other than solvent water into the solventwater, solidifying the obtained solution, and drying the solidifiedmedium to remove water, wherein water is removed in such an amount thatthe solid medium after the removal of water should have the 10minute-average water absorption rate of at least 0.05 ml/minute, and theamount of the solvent water is larger than a prescribed amount by anamount almost equal to the amount of the water to be removed.
 2. Thesolid medium according to claim 1, wherein the amount of the water to beremoved is at least 5% of the solvent water.
 3. The solid mediumaccording to claim 1, wherein the amount of the water to be removed isat least 30% of the solvent water.
 4. The solid medium according to anyone of claims 1 to 3, which has a water content of at least 90%.
 5. Amethod for producing a solid medium comprising the steps of dissolvingcomponents of the solid medium other than solvent water into the solventwater, solidifying the obtained solution and drying the solidifiedmedium to remove water, wherein water is removed in such an amount thatthe solid medium after the removal of water should have a 10minute-average water absorption rate of at least 0.05 ml/minute, and theamount of solvent water is larger than an prescribed amount by an amountalmost equal to the amount of the water to be removed.
 6. The productionmethod according to claim 5, wherein the amount of the water to beremoved is at least 5% of the solvent water.
 7. The production methodaccording to claim 5, wherein the amount of the water to be removed isat least 30% of the solvent water.